This proposal is designed to yield new information on the process of cell division in the bacterium Escherichia coli. Understanding the details of this process is important in designing new classes of antibiotics that can be used in the treatment of bacterial infections. We are currently focusing on two cytoplasmic membrane proteins required for cell division, FtsL and FtsQ. We wish to determine the features of these proteins that cause them to localize to the cell division site. We will obtain a collection of dominant negative mutations in the ftsQ and ftsL genes. Localization of these mutant proteins to mid-cell will be determined with green fluorescent protein (GFP) fusions, using fluorescence microscopy. The dominant-negative mutants will be used to select for mutations that fail to localize to the cell division site. These mutations will help define the region(s) of these proteins that direct them to that site. Mutations in other genes or multi-copy plasmids that suppress the localization defects in FtsQ and FtsL may reveal other proteins these proteins interact with. Multi-copy suppressors of the dominant mutants themselves could reveal the identity of interacting proteins. Protein-protein interactions will be further probed by examining the functioning in E. coli of pairs of proteins from closely-related species of bacteria. A newly discovered gene will provide a test case of this approach. We will use genetic analysis with bioinformatic approaches to help uncover the function of the FtsQ and FtsL proteins. The dominant-negative and other mutations of ftsQ should yield information on the functional regions of the protein. Defining residues important for the activity of this protein would point to particular regions of the protein to study further. We would then continue our computer searches of databases for proteins that share small regions of homology with such regions of FtsQ. In addition, suppressors of an ftsQ null mutant, if obtained, may give a clue as to the protein's function.